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Plasma cell enrichment plays a pivotal role in the accurate prognosis and molecular characterization of multiple myeloma. The separation is commonly carried out by positive cell selection using CD138 monoclonal antibody conjugated to magnetic beads. Optimally, during the separation procedure, the cells should neither be damaged, nor should their phenotype be significantly altered, as these changes would falsify the results if the isolated cells were subsequently used. For this reason, we investigated the expression patterns of different surface markers by flow cytometry before and after magnetic isolation using bone marrow or peripheral blood samples from 12 patients with plasma cell disorders. The selected markers are not only used as backbone markers in routine diagnostics (CD19, CD38, CD45, CD117, and CD138), but they also play an important role in cell adhesion and connection with microenvironment (CD44, CD49d, CD56, and CD81) or possibly drug resistance (CD69, CD86, and CD184), making them promising targets for myeloma research. Moreover, we examined the effects of separation on cell viability in 8 cases. The intensities of 8 out of the 12 investigated markers were slightly influenced, while CD138, CD38, CD56, and CD184 were changed significantly, however the immunophenotype of the cells was not changed. Positive markers remained positive and negative ones remained negative after the separation procedure. In addition, the number of apoptotic plasma cells was significantly reduced during separation, facilitating further examination of the cells. Our results showed that magnetic isolation can be considered as a reliable option but the immunophenotype of plasma cells should be validated after the separation if the intensities of the markers are important for further experiments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Czeti, Sashalmi, Takács, Szalóki, Kriston, Varga, Farkas, Hamed, Márk and Barna.)

Objectives: CD49f is an adhesion molecule present on malignant lymphoblasts in B-cell acute lymphoblastic leukemia; it is associated with a poor prognosis. CD49f expression has been proposed as a marker for measurable residual disease (MRD) marker, but this marker has yet to be implemented in clinical practice., Methods: In this study, we used flow cytometry to detect CD49f expression by leukemic blasts in paired bone marrow and cerebrospinal fluid samples at diagnosis and bone marrow at day 15 of treatment., Results: At diagnosis, 93% of bone marrow and 100% of cerebrospinal fluid lymphoblasts expressed CD49f. The intensity of CD49f expression statistically significantly increased during treatment (P < .001). In MRD-negative end-of-treatment samples, only a small population of hematogones expressed CD49f. Interestingly, the intensity of CD49f expression varied among the different groups of recurrent genetic abnormalities. The ETV6::RUNX1 fusion and ETV6::RUNX1 combined with the high hyperdiploid group were associated with increased expression, whereas the Philadelphia-like group showed low CD49f expression. The lower CD49f expression at diagnosis predicted a lower MRD rate at day 15 of treatment., Conclusions: We concluded that CD49f can be used as an MRD marker and possible prognostic factor in B-cell acute lymphoblastic leukemia., (© American Society for Clinical Pathology, 2024.)

In the pathogenesis of chronic lymphocytic leukemia (CLL) the microenvironment plays an important role, as it produces survival signals and mediates drug resistance. Lenalidomide, which has immunomodulatory effect, can enhance the activation of T-, NK-cells and endothelial cells, however there are no data available whether it can modulate bone marrow stromal cells (BMSCs). In our study, we investigated the effects of lenalidomide on BMSCs and CLL cells. CLL cells were cultured alone or with BMSCs and were treated with lenalidomide. Apoptosis, immunophenotype, and cytokine secretion of BMSCs and CLL cells were determined by flow cytometry. Lenalidomide slightly increased the apoptosis of CLL cells and abrogated the anti-apoptotic effect of BMSCs on CLL cells. Lenalidomide treatment decreased the expression of antigens on CLL cells, which mediate the interactions with the microenvironment. Interestingly, lenalidomide enhanced the expression of IRF4 and the co-stimulatory molecule CD86. The secretion of several cytokines was not changed significantly by lenalidomide. CD49d-negative CLL cases were more sensitive to lenalidomide treatment. Our results suggest that lenalidomide has a limited effect on BMSCs, but it renders CLL cells more immunogenic and unresponsive to survival signals provided by BMSCs., (© 2021 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.)

Up-regulation of the long non-coding RNA LINC00152 can contribute to cancer development, proliferation and invasion, including colorectal cancer, however, its mechanism of action in colorectal carcinogenesis and progression is only insufficiently understood. In this work we correlated LINC00152 expression with promoter DNA methylation changes in colorectal tissues along the normal-adenoma-carcinoma sequence and studied the effects of LINC00152 silencing on the cell cycle regulation and on the whole transcriptome in colon carcinoma cells using cell and molecular biology techniques. LINC00152 was significantly up-regulated in adenoma and colorectal cancer (p < 0.001) compared to normal samples, which was confirmed by real-time PCR and in situ hybridization. LINC00152 promoter hypomethylation detected in colorectal cancer (p < 0.01) was strongly correlated with increased LINC00152 expression (r=-0.90). Silencing of LINC00152 significantly suppressed cell growth, induced apoptosis and decreased cyclin D1 expression (p < 0.05). Whole transcriptome analysis of LINC00152-silenced cells revealed significant down-regulation of oncogenic and metastasis promoting genes (e.g. YES proto-oncogene 1, PORCN porcupine O-acyltransferase), and up-regulation of tumour suppressor genes (e.g. DKK1 dickkopf WNT signalling pathway inhibitor 1, PERP p53 apoptosis effector) (adjusted p < 0.05). Pathway analysis confirmed the LINC00152-related activation of oncogenic molecular pathways including those driven by PI3K/Akt, Ras, WNT, TP53, Notch and ErbB. Our results suggest that promoter hypomethylation related overexpression of LINC00152 can contribute to the pathogenesis of colorectal cancer by facilitating cell progression through the up-regulation of several oncogenic and metastasis promoting pathway elements.

Micro-environment plays important role in the pathogenesis of CLL by providing protective niche for CLL cells. Several molecules play important role in communication between CLL cells and immune cells like CD86.Some of the data suggest that CLL patients with high CD86 level need earlier treatments and cells with higher CD86 expression has higher proliferation rate but the role of CD86 in the survival and proliferation of CLL cells is unclear. We investigated the effect of CD86 expression to CLL cells in 50 peripheral blood and 15 lymph node biopsy samples from CLL patients. Our results showed that the expressions of CD86 increased significantly after 7 day culturing in medium, or in the presence of bone marrow stromal cells (BMSCs). We found positive correlation between CD86 and CD23 expression (p < 0.05), but no correlation with other markers. Furthermore, no correlation were found between the CD86 expression and the proliferation of CLL cells. Analysis of clinical data showed that cases with high CD86 expression had lower level of serum lymphocyte count (p < 0.04) at the time of the diagnosis. CD86 shows multiple appearances in the lymph nodes containing pseudofollicules, but no correlation was found between CD86 positivity, and Ki67 positivity. Our results suggest that the use of CD86 molecule as a proliferation marker for CLL is highly questionable. However, the CD86 molecule may interfere with the immune system of patients with CLL by activating and depleting immune functions. That can be the reason why CD86 positivity may mean worse prognosis.

CD49d and CXCR4 are key determinants of interactions between chronic lymphocytic leukemia (CLL) tumor cells and their microenvironment. In this study, we investigated the effect of CD49d and CXCR4 expressions on survival of CLL cells. Primary CLL cells were cultured with CD49d ligand, VCAM-1, or bone marrow stromal cells (BMSCs); then, apoptosis and immunophenotype analyses were performed. VCAM-1 treatment could not induce direct apoptosis protection or immunophenotype change on the CD49d-expressing CLL cells, but resulted in actin reorganization. The BMSC-induced apoptosis protection was independent from the presence of CD49d expression of CLL cells, but showed an inverse correlation with their CXCR4 expression level. We suppose that CD49d contributes to enhanced survival of leukemic cells by mediating migration to the protective microenvironment, not by direct prevention of apoptosis. Moreover, CLL cells with low CXCR4 expression represent a subpopulation that is more dependent on the microenvironmental stimuli for survival, and show increased "death by neglect" when separated from the supportive niche.

The primary aim of this study was to determine mTOR-pathway activity in primary central nervous system lymphoma (PCNSL), which could be a potential target for therapy. After demonstrating that p-S6 positivity largely exceeded mTOR activity, we aimed to identify other pathways that may lead to S6 phosphorylation. We measured mTOR activity with immunohistochemistry for p-mTOR and its downstream effectors p(T389)-p70S6K1, p-S6, and p-4E-BP1 in 31 cases of PCNSL and 51 cases of systemic diffuse large B-cell lymphoma (DLBCL) and evaluated alternative S6 phosphorylation pathways with p-RSK, p(T229)-p70S6K1, and PASK antibodies. Finally, we examined the impact of PASK inhibition on S6 phosphorylation on BHD1 cell line. mTOR-pathway activity was significantly less frequent in PCNSL compared with DLBCL. p-S6 positivity was related to mTOR-pathway in DLBCL, but not in PCNSL. Among the other kinases potentially responsible for S6 phosphorylation, PASK proved to be positive in all cases of PCNSL and DLBCL. Inhibition of PASK resulted in reduced expression of p-S6 in BHD1-cells. This is the first study demonstrating an mTOR independent p-S6 activity in PCNSL and that PASK may contribute to the phosphorylation of S6. Our findings also suggest a potential role of PASK in the pathomechanism of PCNSL and in DLBCL.

Microglia are highly dynamic cells in the brain. Their functional diversity and phenotypic versatility brought microglial energy metabolism into the focus of research. Although it is known that microenvironmental cues shape microglial phenotype, their bioenergetic response to local nutrient availability remains unclear. In the present study effects of energy substrates on the oxidative and glycolytic metabolism of primary - and BV-2 microglial cells were investigated. Cellular oxygen consumption, glycolytic activity, the levels of intracellular ATP/ADP, autophagy, mTOR phosphorylation, apoptosis and cell viability were measured in the absence of nutrients or in the presence of physiological energy substrates: glutamine, glucose, lactate, pyruvate or ketone bodies. All of the oxidative energy metabolites increased the rate of basal and maximal respiration. However, the addition of glucose decreased microglial oxidative metabolism and glycolytic activity was enhanced. Increased ATP/ADP ratio and cell viability, activation of the mTOR and reduction of autophagic activity were observed in glutamine-supplemented media. Moreover, moderate and transient oxidation of ketone bodies was highly enhanced by glutamine, suggesting that anaplerosis of the TCA-cycle could stimulate ketone body oxidation. It is concluded that microglia show high metabolic plasticity and utilize a wide range of substrates. Among them glutamine is the most efficient metabolite. To our knowledge these data provide the first account of microglial direct metabolic response to nutrients under short-term starvation and demonstrate that microglia exhibit versatile metabolic machinery. Our finding that microglia have a distinct bioenergetic profile provides a critical foundation for specifying microglial contributions to brain energy metabolism., (Copyright © 2017. Published by Elsevier B.V.)

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of monoclonal plasma cells (PCs) in the bone marrow and other tissues. Although there are several new therapies, MM remains fatal. The interaction between MM cells and the bone marrow microenvironment promotes drug resistance and cancer cells survival. In our present work, we compared the antigen expression pattern of normal and pathological PCs and investigated the possible connections between various surface receptors, adhesion molecules, and recurrent genetic aberrations. We showed that the expression of CD29, CD27, and CD81 is lower in MM cells than in normal PCs. We found correlation of chromosome 11 hyperdiploidity and the decrease of CD27 expression. We demonstrated that MM cells with CD20 positivity also have CD28 expression. Multiple myeloma patients with active CD29 showed better response to treatment. Our results suggest that these changes may result in an alteration of the interaction between stromal cell and MM cell facilitating cell survival and the development of a more aggressive and resistant phenotype., (Copyright © 2016 John Wiley & Sons, Ltd.)

Background: Multiple studies concluded that oncometabolites (e.g. D-2-hydroxyglutarate (2-HG) related to mutant isocitrate dehydrogenase 1/2 (IDH1/2) and lactate) have tumour promoting potential. Regulatory mechanisms implicated in the maintenance of oncometabolite production have great interest. mTOR (mammalian target of rapamycin) orchestrates different pathways, influences cellular growth and metabolism. Considering hyperactivation of mTOR in several malignancies, the question has been addressed whether mTOR operates through controlling of oncometabolite accumulation in metabolic reprogramming., Methods: HT-1080 cells - carrying originally endogenous IDH1 mutation - were used in vitro and in vivo. Anti-tumour effects of rapamycin were studied using different assays. The main sources and productions of the oncometabolites (2-HG and lactate) were analysed by 13 C-labeled substrates. Alterations at protein and metabolite levels were followed by Western blot, flow cytometry, immunohistochemistry and liquid chromatography mass spectrometry using rapamycin, PP242 and different glutaminase inhibitors, as well., Results: Rapamycin (mTORC1 inhibitor) inhibited proliferation, migration and altered the metabolic activity of IDH1 mutant HT-1080 cells. Rapamycin reduced the level of 2-HG sourced mainly from glutamine and glucose derived lactate which correlated to the decreased incorporation of 13 C atoms from 13 C-substrates. Additionally, decreased expressions of lactate dehydrogenase A and glutaminase were also observed both in vitro and in vivo., Conclusions: Considering the role of lactate and 2-HG in regulatory network and in metabolic symbiosis it could be assumed that mTOR inhibitors have additional effects besides their anti-proliferative effects in tumours with glycolytic phenotype, especially in case of IDH1 mutation (e.g. acute myeloid leukemias, gliomas, chondrosarcomas). Based on our new results, we suggest targeting mTOR activity depending on the metabolic and besides molecular genetic phenotype of tumours to increase the success of therapies.

Chronic lymphocytic leukemia (CLL) is characterized by a neoplastic B-cell population coexpressing CD5 and CD23; however, the expression of CD23 is variable. In human, two isotypes of CD23 have been identified and related to different functions. The aim of our study was to investigate the relative expression of the two CD23 isotypes in CLL and find possible correlation with other prognostic factors. The expression of CD23 isotypes was analyzed in 54 cases of CLL by polymerase chain reaction (PCR) and quantitative real-time PCR. The immunophenotype of CLL cells was characterized by flow cytometry. We demonstrated a higher CD23a than CD23b expression of CLL cells. Our results also revealed two subsets of CLL cases with a distinct CD23 isotype expression pattern. Thirty-two percent of the cases (group CLL1) showed both low mRNA level of CD23 isotypes and high protein levels of CD20 and CD38 in contrast to group CLL2 with high CD23 mRNA levels. By correlating these results to the presence of prognostic factors determined by fluorescence in situ hybridization, we found that the majority of the cases of group CLL1 (14/17) carried trisomy 12. In summary, our results confirm a high CD23a/CD23b ratio of the CLL cells and demonstrate that in a subset of CLL cases, low CD23 expression together with high CD20 and CD38 expressions may serve as a surrogate for trisomy 12. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Background: Alterations in cellular metabolism are considered as hallmarks of cancers, however, to recognize these alterations and understand their mechanisms appropriate techniques are required. Our hypothesis was to determine whether dominant bioenergetic mechanism may be estimated by comparing the substrate utilisation with different methods to detect the labelled carbon incorporation and their application in tumour cells., Methods: To define the bioenergetic pathways different metabolic tests were applied: (a) measuring CO2 production from [1-(14)C]-glucose and [1-(14)C]-acetate; (b) studying the effect of glucose and acetate on adenylate energy charge; (c) analysing glycolytic and TCA cycle metabolites and the number of incorporated (13)C atoms after [U-(13)C]-glucose/[2-(13)C]-acetate labelling. Based on [1-(14)C]-substrate oxidation two selected cell lines out of seven were analysed in details, in which the highest difference was detected at their substrate utilization. To elucidate the relevance of metabolic characterisation the expression of certain regulatory factors, bioenergetic enzymes, mammalian target of rapamycin (mTOR) complexes (C1/C2) and related targets as important elements at the crossroad of cellular signalling network were also investigated., Results: Both [U-(13)C]-glucose and [1-(14)C]-substrate labelling indicated high glycolytic capacity of tumour cells. However, the ratio of certain (13)C-labelled metabolites showed detailed metabolic differences in the two selected cell lines in further characterisation. The detected differences of GAPDH, β-F1-ATP-ase expression and adenylate energy charge in HT-1080 and ZR-75.1 tumour cells also confirmed the altered metabolism. Moreover, the highly limited labelling of citrate by [2-(13)C]-acetate-representing a novel functional test in malignant cells-confirmed the defect of TCA cycle of HT-1080 in contrast to ZR-75.1 cells. Noteworthy, the impaired TCA cycle in HT-1080 cells were associated with high mTORC1 activity, negligible protein level and activity of mTORC2, high expression of interleukin-1β, interleukin-6 and heme oxygenase-1 which may contribute to the compensatory mechanism of TCA deficiency., Conclusions: The applied methods of energy substrate utilisation and other measurements represent simple assay system using (13)C-acetate and glucose to recognize dominant bioenergetic pathways in tumour cells. These may offer a possibility to characterise metabolic subtypes of human tumours and provide guidelines to find biomarkers for prediction and development of new metabolism related targets in personalized therapy.